Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

Summary

This method estimates sediment denitrification rates in sediment cores using the acetylene inhibition technique and microsensor measurements of the accumulated N₂O. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N₂O accumulation, and calculating the denitrification rate.

Abstract

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has become particularly relevant for assessing the anthropogenic-altered global nitrogen cycle and the emission of greenhouse gases (i.e., N₂O). Several methods are available for measuring denitrification, but none of them are completely satisfactory. Problems with existing methods include their insufficient sensitivity, and the need to modify the substrate levels or alter the physical configuration of the process using disturbed samples.This work describes a method for estimating sediment denitrification rates that combines coring, acetylene inhibition, and microsensor measurements of the accumulated N₂O. The main advantages of this method are a low disturbance of the sediment structure and the collection of a continuous record of N₂O accumulation; these enable estimates of reliable denitrification rates with minimum values up to 0.4-1 µmol N₂O m^-2 h^-1. The ability to manipulate key factors is an additional advantage for obtaining experimental insights. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N₂O accumulation, and calculating the denitrification rate. The method is appropriate for estimating denitrification rates in any aquatic system with retrievable sediment cores. If the N₂O concentration is above the detection limit of the sensor, the acetylene inhibition step can be omitted to estimate the N₂O emission instead of denitrification. We show how to estimate both actual and potential denitrification rates by increasing nitrate availability as well as the temperature dependence of the process. We illustrate the procedure using mountain lake sediments and discuss the advantages and weaknesses of the technique compared to other methods. This method can be modified for particular purposes; for instance, it can be combined with ¹⁵N tracers to assess nitrification and denitrification or field in situ measurements of denitrification rates.

Introduction

Anthropogenic alteration of the nitrogen cycle is one of the most challenging problems for the Earth system¹. Human activity has at least doubled the levels of reactive nitrogen available to the biosphere². However, there remain large uncertainties regarding how the global N cycle is evaluated. A few flux estimates have been quantified with less than ±20% error, and many have uncertainties of ±50% and larger³. These uncertainties indicate the need for accurate estimations of denitrification rates across ecosystems and an understanding of the underlying mechanisms of variation. Denitrification is a....

Protocol

1. Preparation

NOTE: Begin this on the day before the measurements are taken.

1. Assemble the measurement setup (Figure 1a, see the Table of Materials).
   NOTE: To ensure a constant and high-quality power supply, the measurement device is connected to the grip via an uninterruptible power supply (UPS) that can also act as a backup. In the case of a long-duration power failure, a car battery serve as an extra power source.
2. Start the sensor's software and apply a -0.8 V voltage to polarize the N₂O microsensors. The ....

Results

A total of 468 denitrification rates were estimated using the protocol above in sediments from Pyrenean mountain lakes over the period 2013-2014. We show some of these results to illustrate the procedure (Figure 2 and Figure 3). In general, the linear model between the N₂O concentration and time has good correlation (R² ≥ 0.9). The slope of the relationship provides an estimate of the denitrification rate (s.......

Discussion

The main advantages of the described method are the use of minimally disturbed sediment core samples and the continuous recording of the N₂O accumulation. These allow estimation of relatively low denitrification rates that are likely similar to those occurring in situ. Nonetheless, some aspects concerning the coring, sensor performance, and potential improvements are discussed.

An apparently simple but critical step of the method is good core recovery. The sediment/water in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Messenger-adapted gravity corer	-	-	Reference in the manuscript. Made by Glew, J.
Sampling tube	-	-	Acrylic. Dimensions: 100 cm (h) × 6.35 cm (d) × 6.50 cm (D). Sharpen the edge of the sampling tube that penetrates into the sediment to minimize the disturbance in the recovered sediment core sample.
Handheld sounder	Plastimo	38074	Echotest II Depth Sounder.
Rubber stopper	VWR	DENE1012114	With two holes, used to mix the N2O-water in the calibration chamber. Dimensions: 20 mm (h) × 14 mm (d) × 18 mm (D) (3 mm hole (D)).
Rubber stopper	VWR	217-0125	To seal the bottom part of the methacrylate tube and to sample in shallow water bodies. Dimensions: 45 mm (h) × 56 mm (d) × 65 mm (D).
Rubber stopper	VWR	217-0126	Place the rubber stopper in the top side of the sampling tube to obtain a vacuum for sampling in littoral zones and shallow water bodies. Dimensions: 50 mm (h) x 60 mm (d) x 70 mm (D).
PVC cover	-	-	To seal the top side part of the acrylic tube. Dimensions: 45 mm (h) × 56 mm (d) × 65 mm (D). Dimensions: 65 mm (D).
Adhesive tape	-	-	Waterproof. To ensure all joints (PVC cover sampling tube and PVC cover sensor) and to avoid water leaks.
Thermometer	-	-	Portable and waterproof, to measure the temperature in the water overlying the sediment just after sampling the cores.
GPS	-	-	To save the location of a new sampling site or to arrive at a previous site.
Wader	-	-	For littoral or shallow site samplings.
Boat	-	-	An inflatable boat is the best option for its lightness if the sampling site is not accessible by car.
Rope	-	-	Rope with marks showing its length (e.g., marked with a color code to distinguish each meter).
N2O gas bottle and pressure reducer	Abelló Linde	32768-100	Gas bottle reference.
C2H2 gas bottle and pressure reducer	Abelló Linde	32468-100	Gas bottle reference.
Tube used to evacuate the excess of water	-	-	Consists of a solid part (e.g., a 5 ml pipette tip without its narrowest end) inserted in a silicone tube.
Nitrous Oxide Minisensor w/ Cap	Unisense	N2O-R	We use 4 sensors at a time.
Microsensor multimeter 4 Ch. 4 pA channels	Unisense	Multimeter	Picoammeter logged to a laptop. The standard device allows for 2 sensor picoammeter connections (e.g., N2O sensor), one pH/mV and a thermometer. We ordered a device with four picoammeter connections, allowing the use of 4 N2O sensors simultaneously.
SensorTrace Basic 3.0 Windows software	Unisense		Sensor data acquisition software.
Calibration Chamber incl. pump	Unisense	CAL300	Calibration chamber. We tuned it with rubber stoppers and syringes to mix the N2O-water without making bubbles.
Incubation chamber	Ibercex	E-600-BV	Indispensable equipment for working at a constant temperature (±0.3 °C). It also allows control of the photoperiod.
Electric stirrer	-	-	Part of the stirring system. It hangs in the water, overlying the sediment subject, by a fishing line that is hooked to the PVC cover.
Electromagnet	-	-	Part of the stirring system. It is fixed to the outside of the acrylic tube, approximately at the same level as the stirrer. It is activated episodically (ca. 1 on-off per s) by a circuit, attracting the stirrer when it is on and releasing it when it is off, thereby generating the movement that agitates the water.
Electromagnetic pulse circuit	-	-	Part of the stirring system. It is connected by wires to the electromagnet and sends pulses of current that turn the electromagnet on and off.
Uninterruptible power supply (UPS)	-	-	It improves the quality of the electrical energy that reaches the measurement device, filtering the highs and low of the voltage, thereby ensuring a more constant and stable N2O sensor signal.